Infectious proteins (prions), originally postulated to explain a set of transmissible spongiform encephalopathies (TSEs), are now known to underlie a number of epigenetic elements in fungi (including the yeast [PSI+] and [URE3] states) and perhaps in higher organisms. Although mammalian PrP and fungal prion proteins are unrelated in amino acid sequence, their prion states share common structural features that place them in a class of misfolded proteins (amyloids) responsible for a broad range of noninfectious diseases. The facile genetics of yeast together with the ability to create de novo, infectious forms of proteins from pure material have greatly aided our understanding of the principles of prion inheritance as well as the role of cellular factors in facilitating and inhibiting prion replication. Much remains to be learned about the specific role of cellular cofactors. Nonetheless, mechanistic parallels between the mammalian and yeast prion phenomena point to universal features of conformation-based infection and inheritance involving propagation of ordered beta-sheet-rich amyloid protein aggregates. These phenomena include the ubiquitous presence of transmission barriers, which inhibit transmission between even closely related prion proteins, and prion strains wherein infectious particles composed of the same protein give rise to a range of different heritable prion states. The goal of the present project is to take advantage of the facile nature of yeast systems together with the ability to reconstitute [PSI+] prion formation in vitro to elucidate the mechanisms of these universal features of prion biology in terms of the structural, biophysical and biochemical properties of beta-sheet-rich protein aggregates and the cellular factors that act on such species. Finally, we anticipate that many of the methodological, experimental, and theoretical advances we are making in studying the mechanisms by which proteins misfold into prion conformations will be directly applicable to related studies on mammalian PrP. To accomplish the above goals, we will focus on the following three Specific Aims: (1) Mechanism of prion growth and replication. We are defining the mechanism by which Sup35 prion fibers capture and catalyze the conversion of normal soluble forms of Sup35. We will also reconstitute and mechanistically analyze the process by which cellular factors such as molecular chaperones sever preformed Sup35 fibers, thereby allowing replication of the prion element. (2) Dissection and design of prion elements. We will extend the dissection approach we used successfully to define the modular architecture of the Sup35 and New1 prion domains to explore other known fungal prions including those of Ure2, Rnq1 and Het-s. We will then use the principles elucidated by these studies to create novel prion elements in yeast including ones based on Abeta, alpha-synuclein and mammalian PrP. (3) Analysis of the structural basis of prion strains. We will use a comprehensive approach combining cryoelectron microscopy (cryo-EM), site-directed spin labeling (SDSL) in conjunction with electron paramagnetic resonance (EPR) spectroscopy together with modeling and mutational studies to define the quaternary and/or tertiary structural differences responsible for the different prion strains.